Tissuefax microscope

Mice were anesthetized with ketamine (10 mg/ml) and were then perfused with 10 mM phosphate-buffered saline (PBS; pH 7.4), followed by 4% (w/v) formaldehyde in PBS. Whole brains were removed, fixed by immersion in 4% (w/v) formaldehyde in 10 mM PBS overnight at 4 °C, and subsequently cryoprotected in 30% (w/v) sucrose in 10 mM PBS. Tissue samples were embedded in paraffin wax and sections cut (4–5 μm) for staining with H&E [24 (link)]. Tissue H&E staining was examined using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), and images were scanned by a TissueFax microscope (TissueGnostics, Vienna, Austria). The area of cerebellar lobules was quantified, the nuclei (stained with hematoxylin) were counted, and the cytoplasm (stained with eosin) in lobules I–V, VI–VII, VIII–IX, and X–XI was quantified using HistoQuest tissue analysis software (TissueGnostics). The angularity of the cerebellum was calculated from the angles from the top of the cerebellum to each lobe and was indicated by angles (A)_1–4, according to the equation [(A₃/A₄) / (A₂/A₁)] × 100%.

For immunohistochemical staining, dewaxed slides were incubated with Peroxidase Block (3% H₂O₂) for 20 min and heated at 95 °C for 20 min in sodium citrate buffer (pH 6.0). After washing and blocking, the slides were incubated with the indicated antibodies against LacZ, caspase 3, Slc7a11, or Atoh-1 for 2 h and then incubated with the indicated mouse/rabbit probe horseradish peroxidase (HRP)-conjugated antibodies (BioTnA, Kaohsiung, Taiwan) for 30 min. After washing, the chromogen was developed by 3,3′-diaminobenzidine staining and the samples were counterstained with haematoxylin. The slides were rinsed with H₂O and covered with resin-based mounting medium (BioTnA) after dehydration. Histopathological analysis was performed by the RIKEN BRC Experimental Animal Division in Japan and the NLAC, Taiwan. Tissue HE-staining was visualized using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), and images were scanned and saved using a TissueFax microscope (TissueGnostics, Vienna, Austria).

Unstained 5 μm-thick TMA sections were deparaffinized and rehydrated using standard methods. IHC was performed on TMA sections using a custom polyclonal antibody specific for the C-terminus of ERβ2 (482-MKMETLLPEATMEQ-495) as previously reported [29 (link)]. ERβ2 stained slides were scanned for pathologist visual scoring using an automated Tissuefax microscope (TissueGnostics, Tarzana, CA) and reviewed via an online web gallery. ERβ2 immunostaining within malignant cells was scored for each TMA spot by a pathologist (X.Z.) blinded to clinical parameters. Cytoplasm and nuclei were evaluated separately. As described previously, immunostaining was assessed using a score calculated by multiplying staining intensity (0 for no staining, 1 for light/weak staining, and 2 for strong/intense staining) by the corresponding percentage of cells staining positive at each intensity (totaling to 100 %) [27 (link)]. Tissue spots that were missing, damaged, contained staining artifacts, or had uncertain histology were excluded from the analysis. Raw data for pathologist visual scores are included in Additional file 1.